Immunoassays, which take advantage of natural immunological reactions, have found wide-spread use as analytical techniques in clinical chemistry. Because of the specificity of the reactions, they are particularly advantageous in quantifying biological analytes that are present in very low concentration in biological fluids. Such analytes (called ligands herein) include, for example, antibodies, therapeutic drugs, narcotics, enzymes, hormones, proteins, etc.
In competitive binding immunoassays, a labeled hapten analogue is placed in competition with unlabeled haptens for reaction with a fixed amount of the appropriate antibody. Unknown concentrations of the hapten can be determined from the measured signal of either the bound or unbound (i.e. free) labeled hapten analogue.
Conventional labels include radioactive tags, enzymes, chromophores, fluorophores, stable free radicals, and enzyme cofactors, inhibitors and allosteric effectors.
In competitive immunoassays for thyronines, such as thyroxine, specific requirements for labeled thyronine hapten analogues (hereafter sometimes LDH) include: 1) at least 60% of the LDH can be bound by excess immobilized antibody; 2) affinity of the LDH for immobilized antibody is such that competition of a fixed amount of LDH with the drug occurs in a therapeutically relevant concentration range; 3) the process used to prepare the LDH requires only a few steps and 4) stability of the LDH against hydrolysis of its enzyme label under storage conditions. Requirements imposed on the thyronine hapten analogues include: 1) accessibility of the analogue to the immobilized antibody following conjugation with the enzyme label; 2) specific recognition of the labeled analogue by the antibody to the drug; and 3) sufficient reactivity of the drug analogue with the enzyme label, either directly or following activation of the enzyme or analogue, under conditions that do not adversely affect enzyme activity.
U.S. Pat. No. 4,786,591 discloses haptens comprising a thyroxine (T4) nucleus having an active ester group. The problem is that these haptens do not provide labeled hapten analogues that are at least 60% immunoreactive.